CN105407913A - Biomarkers for identifying esophageal cancer patients for treatment with an anti-EGFR drug - Google Patents

Abstract

The present invention provides methods for the treatment of esophageal cancer patients as well as the identification and selection of esophageal cancer patients for treatment with an anti-EGFR agent treatment including anti-EGFR antibody treatment, e.g., cetuximab treatment. In some embodiments, the drug is against a heterodimer formed by EGFR and another member of the ErbB receptor family such as EfbB2/Her2/neu.

Description

For the identification of the biomarker of the patient with esophageal carcinoma of available anti-EGFR Drug therapy

The cross reference of related application

This application claims the priority of the international patent application no PCT/CN2012/079411 that on July 31st, 2012 submits to, it is incorporated to its entirety by carrying stating at this.

Invention field

The present invention relates to patient with esophageal carcinoma treatment and for using for the treatment of the medicine of EGF-R ELISA (EGFR), as anti-egfr antibody therapy, such as, Cetuximab (cetuximab), the qualification of patient with esophageal carcinoma and selection.

Background of invention

The esophageal carcinoma (ESC) is one of the most fatal cancer, and it has 5 years survival rates (the national esophageal-gastric cancer examination: in the examination of England and Wales to the nursing that the personage suffering from esophageal-gastric cancer obtains lower than 10%.Annual report London for the third time, NHS information centre; 2010).Existing two kinds of main ESC histological types: squamous cell carcinoma (SCC) and adenocarcinoma (ADC).Except operation, the therapeutic choice of standard is chemoluminescence therapy (CRT).All these has limited effect.Esophagus SCC is popular especially in some areas of China, and does not have effective targeted therapies.This area exists the treatment of the esophageal carcinoma to be needed.

Present invention accomplishes these needs and provide the method for effectively treating the esophageal carcinoma.

Brief summary of the invention

The invention provides the method for being treated the esophageal carcinoma in patients by anti-epidermal growth factor receptor (EGFR) agent.

In some embodiments, described method comprises anti-EGFR agent patient being used to effective dose.In some embodiments, the direct targeting EGFR of described medicine.In some embodiments, the downstream signaling pathway of described drug targeting EGFR.In some embodiments, described medicine is antagonist or the antibody of EGFR part, for example, the antagonist of epidermal growth factor (EGF), transforming growth factor α (TGF α), HB-EGF, amphiregulin (amphiregulin), β cytokines, epigen and/or epiregulin or antibody.In some embodiments, described medicine is micromolecule.In some embodiments, the heterodimer that formed as EfbB2/Her2/neu for other members by EGFR and ErbB receptor family of described medicine.In some embodiments, described medicine is for the homodimer formed by EGFR.In some embodiments, described medicine is anti-EGFR agent, and it comprises anti-egfr antibodies therapy, such as, and Cetuximab.

Invention further provides the method with anti-EGFR agent treatment, wherein with the patient that anti-EGFR agent carries out treating, there is one or more EGFR biomarkers.In some embodiments, described anti-EGFR agent comprises anti-egfr antibodies.In some embodiments, described anti-egfr antibodies comprises Cetuximab.

The present invention also carries has encircleed for detecting one or more EGFR biomarker presence or absence and treating patient when one or more EGFR biomarkers exist with anti-EGFR agent.In some embodiments, described anti-EGFR agent comprises anti-egfr antibodies.In some embodiments, described anti-egfr antibodies comprises Cetuximab.

The present invention further provides for the identification of the anti-respondent of EGFR agent therapy and/or the method for non-responder patient.In some embodiments, described anti-EGFR agent comprises anti-egfr antibodies.In some embodiments, described anti-egfr antibodies comprises Cetuximab.Respondent and non-responder patient are identified by the presence or absence detecting one or more EGFR biomarkers in the biological sample obtained from the patient suffering from the esophageal carcinoma.According to this method, the existence of one or more EGFR biomarkers indicates the respondent of anti-EGFR agent therapy, and one or more EGFR biomarkers does not exist the non-responder indicating anti-EGFR agent therapy.

Present invention also offers the method for determining the therapeutic scheme being used for the treatment of the esophageal carcinoma in patients.In this respect, the presence or absence of one or more EGFR biomarkers is detected in the biological sample that described method is included in from the patient suffering from the esophageal carcinoma.The existence of one or more EGFR biomarkers indicates described patient to be the respondent of anti-EGFR agent therapy, and EGFR biomarker does not exist the non-responder that the described patient of instruction is anti-EGFR agent therapy.In some embodiments, described anti-EGFR agent comprises anti-egfr antibodies.In some embodiments, described anti-egfr antibodies comprises Cetuximab.When one or more EGFR biomarkers exist, described patient treats by anti-EGFR agent therapy subsequently.

Present invention also offers by detect from accept the nursing of the standard esophageal carcinoma or anti-EGFR agent therapy patient biological sample in the presence or absence of one or more EGFR biomarkers for changing or revise the therapeutic scheme of anti-EGFR agent therapy and changing based on the presence or absence of one or more EGFR biomarkers in described biological sample and the method for amendment therapeutic scheme.In some embodiments, described anti-EGFR agent comprises anti-egfr antibodies.In some embodiments, described anti-egfr antibodies comprises Cetuximab.For example, when one or more EGFR biomarkers exist, continue anti-EGFR agent therapy, and when one or more EGFR biomarkers do not exist, stop anti-EGFR agent therapy.

Invention further provides the method suffering from the patient of the esophageal carcinoma selecting available anti-EGFR agent therapy for treating.These methods comprise detection from one or more EGFR biomarker presence or absence in the biological sample of described patient, wherein the existence of one or more EGFR biomarkers indicates the respondent of anti-EGFR agent therapy, and EGFR biomarker does not exist the non-responder indicating anti-EGFR agent therapy.The patient that there are one or more EGFR biomarkers is selected to be used for the treatment of anti-EGFR agent therapy subsequently.In some embodiments, described anti-EGFR agent comprises anti-egfr antibodies.In some embodiments, described anti-egfr antibodies comprises Cetuximab.

For described method of the present invention, described EGFR biomarker can be the activity of EGFR gene and/or amplification, EGFR expression, EGFRRNA level, activity of EGFR, EGFR pathway activation or EGFR approach intracellular signaling any body in or external indicator, can be when it is compared with matched group and increase or reduce.In one embodiment, EGFR biomarker comprises any type of relevant to the activity of EGFR sudden change at DNA, RNA or protein level, such as, and the activation of EGFR or the amplification of gene.In another embodiment, EGFR biomarker comprises directly or indirectly relevant to activity of EGFR anyly to measure, such as, and the activation of EGFR or the amplification of gene.In another embodiment, EGFR biomarker comprises the two sudden change of L858R/T790M, insertion mutation (extron 20: 2319-2320AACCCCCAC) and deletion mutation (exons 1 9:2236-2350).In another embodiment, EGFR biomarker comprises relevant to activity of EGFR and is having any biomarker under SCC histological esophageal carcinoma background.In some embodiments, described method comprises the protein expression level detecting EGFR.The protein expression level of EGFR is determined by any suitable method known to those skilled in the art.In some embodiments, described protein expression level is determined by immunohistochemistry (IHC), immunoblotting, Western Immuno dyeing, Western Immuno precipitation, immunoelectrophoresis, immunoblotting, BCA algoscopy, spectrophotography, mass spectrography or enzyme assay.

Present invention also offers as determining, assessing or monitor the treatment of the esophageal carcinoma or treat the method that effect provides useful information.Described method is included in the presence or absence determining one or more EGFR biomarkers in the biological sample from patient, and providing one or more EGFR biomarker presence or absence determination results described to entity, described entity provides treatment or determination or the assessment for the treatment of effect based on the presence or absence of one or more EGFR biomarkers.

The present invention also provides test kit.Described test kit comprises reagent for measuring one or more EGFR biomarkers in biological sample and optional for using the result of one or more EGFR biomarkers described to determine the description of the therapy of the esophageal carcinoma.For example, the existence of one or more EGFR biomarkers indicates the respondent of anti-EGFR agent therapy, and EGFR biomarker does not exist the non-responder indicating anti-EGFR agent therapy.

Accompanying drawing is sketched

Fig. 1 shows representational ESC-SCC to the response of Cetuximab.As shown in the figure, mice accepts Cetuximab or the vehicle control (saline) of 1mg.

Fig. 2 shows the pharmacokinetics effect of Cetuximab in ESC-SCC model.Single-dose treatment is carried out with the agent identical with described in Fig. 1.Analyze at the IHC of shown time point results GA022 tumor sample for pERK biomarker: IHC image (A) and IHC mark (B).Representational photo (400x) illustrates with the positive cell core of pERK biomarker in the GAM022 xenotransplantation of single-dose treatment and Cytoplasm dyeing.When the normal rabbit igg that first antibody is used as negative control substitutes, without detectable immunostaining.

Fig. 3 summarises the activity of Cetuximab and the parameter of EGFR.Little figure from top to bottom shows by expressing the EGFRmRNA that is quantitative, that measured by gene microarray analysis of tumor response of measuring of Δ T/ Δ C value and EGFR expresses.

Fig. 4 shows representational fish analysis.Zuo Tu: ES110P5, and right figure: Patient Sample A (PA).

The quantitative little figure of block diagram of Fig. 5, show from top to bottom by Δ T/ Δ C value measure the EGFR that is quantitative, that measured by gene microarray analysis of tumor response is expressed, EGFR gene copy number (PICNIC) and EGFR gene copy number (PennCNV).

Fig. 6 shows to sum up the block diagram of result.Little figure show from top to bottom by Δ T/ Δ C value measure the EGFR that is quantitative, that measured by gene microarray analysis of tumor response is expressed, EGFR gene copy number (PICNIC) and EGFR gene copy number (PennCNV).

Detailed Description Of The Invention

Part of the present invention is the discovery that can be used for treating the esophageal carcinoma based on anti-EGFR agent.In addition, its part also can be used as the biomarker of predictability for determining whether patient can resist the discovery of EGFR agent therapy response based on EGFR biomarker.Correspondingly, the invention provides the method being used for the treatment of the esophageal carcinoma and the method for the identification of the patient with esophageal carcinoma be suitable for anti-EGFR agent therapy for treating.In some embodiments, described drug targeting EGFR.In some embodiments, the signal transduction path in described drug targeting EGFR downstream.In some embodiments, described medicine is antagonist or the antibody of EGFR part, for example, the antagonist of epidermal growth factor (EGF), transforming growth factor α (TGF α), HB-EGF, amphiregulin, β cytokines, epigen and/or epiregulin or antibody.In some embodiments, described medicine is micromolecule.In some embodiments, the heterodimer that formed as EfbB2/Her2/neu for other members by EGFR and ErbB receptor family of described medicine.In some embodiments, described medicine is for the homodimer formed by EGFR.

The invention provides the method for the treatment of the esophageal carcinoma in patients, it comprises anti-EGFR agent therapy patient being used to effective dose.In some embodiments, with the patient of anti-EGFR agent treatment, there is one or more EGFR biomarkers.

In some embodiments, the invention provides the method for the treatment of the esophageal carcinoma in the patient having one or more EGFR biomarkers, it comprises anti-EGFR agent therapy patient being used to effective dose.In some other embodiments, the invention provides the method for the treatment of the esophageal carcinoma in the patient having one or more EGFR biomarkers, it comprises the anti-egfr antibodies therapy described patient being used to effective dose, such as, and Cetuximab.

In some embodiments, the invention provides the method for treating the esophageal carcinoma in patients, it comprises the presence or absence detecting one or more EGFR biomarkers.When one or more EGFR biomarkers exist, by the anti-EGFR agent therapy of effective dose, patient is treated subsequently.

In some embodiments, present invention also offers the method for the identification of respondent and non-responder patient, it is included in the presence or absence detecting one or more EGFR biomarkers in the biological sample from the patient suffering from the esophageal carcinoma.The existence of one or more EGFR biomarkers indicates the respondent of anti-EGFR agent therapy, and one or more EGFR biomarkers does not exist the non-responder indicating anti-EGFR agent therapy.

In some embodiments, the invention provides the method for determining the therapeutic scheme for the treatment of the esophageal carcinoma in patients.These methods are included in the presence or absence from detecting one or more EGFR biomarkers in the biological sample suffering from patient with esophageal carcinoma.The existence of one or more EGFR biomarkers indicates the respondent of anti-EGFR agent therapy, and EGFR biomarker does not exist the non-responder indicating anti-EGFR agent therapy.When one or more EGFR biomarkers exist, described method can comprise further with anti-EGFR agent therapy for treating patient.

In some embodiments, the invention provides the method for the therapeutic scheme for changing anti-EGFR agent therapy.These methods are included in biological sample the presence or absence detecting one or more EGFR biomarkers, and change described therapeutic scheme based on the presence or absence of one or more EGFR biomarkers.When one or more EGFR biomarkers exist, continue anti-EGFR agent therapy and change according to the method that medical domain is known in some cases, and when one or more EGFR biomarkers do not exist, stopping anti-EGFR agent therapy.

In some embodiments, the invention provides the method suffering from the patient of the esophageal carcinoma for selecting with anti-EGFR agent therapy for treating, it is included in the presence or absence detecting one or more EGFR biomarkers in the biological sample from patient.The existence of one or more EGFR biomarkers indicates the respondent of anti-EGFR agent therapy, and EGFR biomarker does not exist the non-responder indicating anti-EGFR agent therapy.Described method is provided as the therapeutic choice that undertaken by anti-EGFR agent therapy further there is the patient of one or more EGFR biomarkers in those.

The existence of one or more EGFR biomarkers indicates the respondent of anti-EGFR agent therapy.In some embodiments, one or more EGFR biomarkers described detected before anti-EGFR agent therapy.In some embodiments, one or more EGFR biomarkers described are detected in the process of anti-EGFR agent therapy.In some embodiments, one or more EGFR biomarkers described are detected after anti-EGFR agent therapy.

Respondent according to the present invention is the individuality showing treatment effect, and non-responder does not represent treatment effect.Phrase " determine treat effect " or " determining the effect of therapy " and change thereof can comprise any for determining that therapy provides the method for benefit for subject.Term " treatment effect " and change thereof generally by indicating with one or more symptom of disease association or the alleviation of symptom, and can be determined by those skilled in the art easily." treatment effect " also can refer to usually and standard or the relevant symptom of toxicity of off-gauge disease treatment (that is, for chemotherapy or the X-ray therapy for the treatment of of cancer) or the prevention of symptom or improvement.The determination for the treatment of effect normally indication and disease specific, and can comprise and known in the art or existingly treat for determining any method patient being provided to beneficial effect.For example, the evidence for the treatment of effect can include but not limited to the alleviation of disease or indication, and for cancer, it can include but not limited to the minimizing of tumor size, neoplasm metastasis or reduction etc.In addition, treatment effect also can comprise the general improvement of experimenter's holistic health, such as but not limited to the increase of the raising of Quality of Life of Patients, experimenter's survival rate of prediction, depressed minimizing or the minimizing (increase of remission time (remissiontime)) of indication relapse rate.(see, such as, Physicians'DeskReference (2010)).

Anti-EGFR agent therapy can comprise any containing one or more treatment increasing, reduce, eliminate, strengthen, postpone, reduce or block the entity of EGFR signal transduction path activity.In some embodiments, described compositions after DNA level, transcriptional level, translation skill, translation level and/or protein level directly for the one or more members in EGFR or EGFR signal transduction path.Described compositions can selectively targeted EGFR or at least targeting EGFR.In some embodiments, described compositions can cause the gene of member in EGFR and/or EGFR signal transduction path to be prevented and/or gene silencing, such as, strikes member that is low or that knock out in EGFR and/or EGFR signal transduction path.In some embodiments, described compositions can modify EGFR protein active, as modified the activity of EGFR and its ligand binding and/or it induces the activity of downstream signaling pathway.In some embodiments, described medicine is antagonist or the antibody of EGFR part, for example, the antagonist of epidermal growth factor (EGF), transforming growth factor α (TGF α), HB-EGF, amphiregulin, β cytokines, epigen and/or epiregulin or antibody.In some embodiments, described medicine can targeting EGFR and/or part block ligand-receptors bind.In some embodiments, described medicine can cause the cellular signal transduction that the conformation change in receptor and/or part also reduces or inactivation EGFR mediates.In some embodiments, the heterodimer that described medicine is formed as EfbB2/Her2/neu for another member by EGFR and ErbB receptor family, or the homodimer formed by two EGFR molecules.EGFR signal transduction path is described in (TargetingtheEGFRsignalingpathwayincancertherapy, ExpertOpinTherTargets, the 2012January such as Sechacharyulu; 16 (1): 15 – 31.), (the Acomprehensivepathwaymapofepidermalgrowthfactorreceptors ignaling such as Oda, MolecularSystemsBiology1:2005.0010), and DevelopmentEGFRSignalingPathway (PathwayMaps, ThomsonReuters, 2012), its each section is all incorporated to for all objects with its entirety herein.

In some embodiments, described agent comprises one or more and such as suppresses at DNA, RNA or protein level or reduce the entity of activity of EGFR.According to the present invention, anti-EGFR agent therapy can comprise any anti-EGFR therapy comprising one or more chemical compounds or compositions, biomolecule and combination thereof.In one embodiment, anti-EGFR agent therapy of the present invention is anti-egfr antibodies therapy.According to the present invention, anti-egfr antibodies therapy can comprise any therapy using anti-egfr antibodies or antibody sample therapeutic agent, and described antibody sample therapeutic agent includes but not limited to any molecule having one or more anti-EGFRCDR.In one embodiment, anti-egfr antibodies therapy comprises any granted anti-egfr antibodies, such as, and Cetuximab (also referred to as Erbitux (erbitux)) or its biological analog or derivant, such as, total man's anti-egfr antibodies etc.Cetuximab (is sold by ImClone and Bristol-MyersSquibb in North America, and sold by MerckKGaA in other regions of the world) be restructuring, people/mouse chimera monoclonal antibody, it blocks the activation of epidermal growth factor (EGF) receptor (EGFR).Cetuximab is used for the treatment of metastatic colorectal cancer and incidence cancer by intravenous infusion administration.In some embodiments, Cetuximab is mixed with the aseptic colourless liquid of pH7.0 to 7.4.In some embodiments, Cetuximab is mixed with 100mg (50mL) or 200mg (100mL) with the concentration of 2mg/mL.In some embodiments, Cetuximab is formulated in the bottle of single use.In some embodiments, described Cetuximab formulation comprises 8.48mg/mL sodium chloride, 1.88mg/mL sodium phosphate dibasic heptahydrate, 0.41mg/mL biphosphate sodium-hydrate and Injectable sterile water.Be that the technical staff of medical domain knows for using the method for Cetuximab and formulation, and anyly know method, the dosage regimen for Cetuximab or the formulation for Cetuximab of using Cetuximab and all can consider jointly to use with method of the present invention.Use the particular combination thing of Cetuximab and method to be described in United States Patent (USP) 8075916,7977336,6217866, its each section is incorporated to for all objects by carrying stating with its entirety.

In some embodiments, described anti-EGFR agent comprises micromolecule.Term used herein " micromolecule " refers to have the molecule being less than 500MW molecular weight, and wherein said medicine is non-peptide or peptide agent.In some embodiments, described pharmaceutical pack is containing protein or polypeptide.In some embodiments, described pharmaceutical pack is containing hybrid molecule.In some embodiments, described medicine is antibody.In some embodiments, described medicine is anti-egfr antibodies.In some embodiments, described medicine is anti-EGFR ligand antibody.In some embodiments, described medicine is humanized anti-EGFR ligand antibody.In some embodiments, described antibody is monoclonal antibody.

In some embodiments, described medicine is anti-egfr antibodies.In some embodiments, described medicine is Cetuximab or its functional variant thereof or derivant.The nonrestrictive embodiment of anti-egfr antibodies is described in PCT publication number WO/2011/140151, WO/2007/058823, WO/2011/080209, WO/2010/080463, WO/2012/020059, WO/2011/080209, WO/2011/059762, WO/2011/152525, WO/2011/140254, WO/2010/034441, WO/2011/156617, WO/2005/090407, WO/2013/006547, WO/2008/140493, WO/2011/156617, U.S. the patent No. 5942602, 6129915, 7723484, 7618631, 7598350 and U.S. patent application publication number 20100166755, 20080274114, 20130142812, 20110158987, 20120107234, 20110117110, 20110287002, 20120149879, 20120282633, 20100009390, 20050238640, 20060154334, 20120231021 and 20130149299, its each section is all incorporated to herein for all objects in full with it.

For method of the present invention, EGFR biomarker can be EGFR gene amplification, EGFR expression, the EGFR of EGFRRNA level, constitutive activity, activity of EGFR, EGFR pathway activation or EGFR approach intracellular signaling in vivo or external any indicator, time compared with matched group, they are whole or increase or reduce.In one embodiment, EGFR biomarker comprises any type of relevant to the activity of EGFR sudden change at DNA, RNA or protein level, and such as, EGFR activates or gene amplification.In another embodiment, EGFR biomarker comprises any direct or indirect measurement relevant to activity of EGFR, and such as, EGFR activates or gene amplification.In some embodiments, EGFR biomarker comprises directly or indirectly relevant to activity of EGFR anyly to measure, such as, and the activation of EGFR or the amplification of gene.In some embodiments, the EGFRRNA level that EGFR biomarker is selected from EGFR gene amplification, the EGFR that increases expresses, increase, the EGFR approach intracellular signaling of the EGFR of constitutive activity and the EGFR pathway activation of enhancing or enhancing.In another embodiment, EGFR biomarker comprises the two sudden change of L858R/T790M, insertion mutation (extron 20: 2319-2320AACCCCCAC), deletion mutation (exons 1 9:2236-2350).In another embodiment, EGFR biomarker comprises relevant to activity of EGFR and is having any biomarker under SCC histological esophageal carcinoma background.

In some embodiments, EGFR gene copy number is the copy number of at least 3,4,5,6,7,8,9,10 or more.In some embodiments, described increase is by comparing with one or more standard level or determining by comparing with the level as standard level known in the art.Measure gene amplification, the expression of increase, the RNA of increase or DNA level and determine that protein be whether the method for constitutive activity is well known, and any such method can adopt jointly with the present invention.

Term used herein " standard level " or " reference level " refer to represent average, the representational feature of one or more biomarker or the standardized data of characteristic or data set in specific population of subjects.These features or characteristic include but not limited to the enzymatic activity etc. of transcript abundance, transcript stability, transcription rate, translation rate, post translational modification, protein abundance, protein stability and/or protein.In some embodiments, described specific population of subjects is made up of the individual subjects of about 5, about 10, about 20, about 50, about 100, about 200, about 300, about 400, about 500, about 1000, about 5000, about 10K or more.In some embodiments, all individual subjects all resist the response of EGFR therapy.In some embodiments, all individual subjects all do not resist the response of EGFR therapy.

In some embodiments, described method comprises and the EGFR biomarker overview (profile) of patient and the EGFR biomarker overview being derived from the standard level resisting the population of subjects that EGFR agent is replied being compared, if wherein the EGFR biomarker overview of patient is within the EGFR biomarker overview of standard level, determine that described experimenter will resist the response of EGFR agent.

In some embodiments, described method comprises and the EGFR biomarker overview (profile) of patient and the EGFR biomarker overview being derived from the standard level resisting the unresponsive population of subjects of EGFR agent being compared, if wherein the EGFR biomarker overview of patient is within the EGFR biomarker overview of standard level, determine that antagonism EGFR agent is not replied by described experimenter.

Statement used herein " the EGFR biomarker overview of patient is within the EGFR biomarker overview of standard level " refers to that the EGFR biomarker overview analyzed is similar to predetermined EGFR biomarker overview, for example, the parameter of patient EGFR biomarker and the parameter similar describing predetermined EGFR biomarker overview are described, or in the excursion of predetermined EGFR biomarker overview, such as, described parameter based on build self-described predetermined EGFR biomarker overview parameter 90% confidence interval excursion within.

EGFR biomarker overview is determined by any appropriate method known to those skilled in the art.In some embodiments, biological sample is taken from experimenter and is analyzed it.In some embodiments, usually subsequently the existence of one or more gene expression products as RNA, mRNA, cDNA, cRNA, protein etc. in described biological sample is detected.

In some embodiments, directly use the mRNA of biological sample to determine the expression of one or more genes by hybridization.In some specific embodiments, RNA obtains from biological sample.Methods known in the art are used described RNA to be transformed into cDNA (complementary DNA) copy subsequently.In some specific embodiments, described cDNA fluorescent labeling or other detectable labellings carry out labelling.Subsequently described cDNA and the substrate comprising multiple interested probe are hybridized.Interested probe is hybridized with at least one DNA sequence of gene signature usually under stringent hybridization condition.In some scheme, described multiple probe can be hybridized with the sequence being derived from gene biological mark under hybridization conditions.In some embodiments, described condition is included in 65 DEG C and uses 6 × SSC (0.9MNaCl, 0.09M sodium citrate, pH7.4).Described probe can comprise nucleic acid.Framework residue or the connection of known nucleotide analog or modification contained in term " nucleic acid ", it can be synthesis, naturally exist exist with non-natural with there is similar binding characteristics with reference to nucleic acid, and it is with the mode metabolism similar to reference nucleotide.The example of these analog includes but not limited to, thiophosphate, phosphoramidate, methyl phosphonate, chiral-methyl phosphonates, peptide-nucleic acid (PNAs).

In some cases, described probe is about 15 to about 50 base pairs or more in length.The amount of cDNA hybridization is measured by testing the existence of detectable labelling as fluorescence.What can use hybridization signal is quantitatively the mark that particular patient or multiple patient generate for the particular sequence in gene signature or sequence sets.

Be included in scope be included under stringent hybridization condition with the DNA array of multiple sequences of one or more biomarker genes sequence hybridizations or microarray.The example comprising the substrate of one or more interested probe is the multiple DNA probes with substrate adhesion.In certain embodiments, described substrate can comprise one or more materials as gel, celluloid, nylon, quartz, glass, metal, silica based materials, silicon dioxide, resin, polymer, etc., and combinations thereof.Usually, described DNA probe comprises the adjoining DNA of about 10-50bp.In certain embodiments, described probe is the adjoining DNA of about 20 to about 50bp.In certain embodiments, the present invention relates to the microarray teachings comprising and use about it.The teachings that described test kit can comprise the container containing one or more microarraies and use about it.

Can use the method that can detect nucleic acid for one or more gene biological marks gene expression analysis described in biological sample, described method includes but not limited to PCR (polymerase chain reaction); RT-PCR (reverse transcriptase-polymerase chain reaction); Quantitatively or semiquantitive PCR, etc.In certain embodiments, the level of gene expression is measured by the protein expression product detecting described gene or DNA sequence.The level of protein can use methods known in the art to measure, and it comprises the antibody used with specified protein specific binding.These antibody, comprise polyclone or monoclonal antibody, and methods known in the art can be used to produce.These antibody also can form antibody chip or Antibody microarray with solid state substrate coupling.Antibody or protein microarray can use methods known in the art to generate.

Any suitable protein detection can be used, the method quantizing and compare, as those are described in Tschesche (MethodsinProteinBiochemistry, ISBNWalterdeGruyter, 2011, ISBN3110252368, 9783110252361), (the Chip-baseddetectionofproteincancermarkers such as Goluch, ProQuest, 2007, ISBN0549463453, 9780549463450), Speicher (ProteomeAnalysis:InterpretingtheGenome, Elsevier, 2004, ISBN0080515304, 9780080515304), (the ProteinArrays such as Albala, BiochipsandProteomics, CRCPress, 2003, ISBN0203911121, 9780203911129), Walker (TheProteinProtocolsHandbook, Springer, 2002, ISBN0896039404, 9780896039407), Fung (ProteinArrays:MethodsandProtocols, Springer, 2004, ISBN1592597599, 9781592597598) and Bienvenut (AccelerationandImprovementofProteinIdentificationbyMassS pectrometry, Springer, 2005, ISBN1402033184, 9781402033186), its each section is incorporated to for all objects with its entirety by carrying stating.In some embodiments, the protein expression level of described biomarker is undertaken detecting and measuring by immunohistochemistry (IHC), immunoblotting, Western Immuno dyeing, Western Immuno precipitation, immunoelectrophoresis, immunoblotting, BCA algoscopy, spectrophotography, mass spectrography or enzyme assay.

For other to the detection of biomarker level, quantification and more relevant method, see, such as, CurrentProtocolsinMolecularBiology, Ed.Ausubel, FrederickM. (2010); CurrentProtocolsinProteinScienceLast, Ed.Coligan, JohnE., wait (2010); CurrentProtocolsinNucleicAcidChemistry, Ed.Egli, Martin (2010); CurrentProtocolsinBioinformatics, Ed.Baxevanis, AndreasD. (2010) and MolecularCloning:ALaboratoryManual, ThirdEdition, Sambrook, Joseph (2001), it is all incorporated to herein with it in full by carrying stating.

For to obtain the method for biological sample be well known and can adopt any method for obtaining biological sample of standard.The biological sample of method of the present invention can be used to include but not limited to serum, blood, blood plasma, whole blood and its derivant, skin, hair, hair follicle, saliva, mouth mucus, vaginal mucus, perspiration, tear, epithelial tissue, urine, sperm (semen), seminal fluid (seminalfluid), refining, prostatic fluid, front ejaculation liquid (examining amber liquid (Cowper'sfluid)), Excreta, biopsy, ascites, cerebrospinal fluid, the sample of lymph and tissue extract or biopsy samples.(see, such as, ClinicalProteomics:MethodsandProtocols, Vol.428inMethodsinMolecularBiology, Ed.AntoniaVlahou (2008) .) in one embodiment, biological sample of the present invention comprises any cell or tissue sample of esophagus, such as, the original position of the esophageal carcinoma or circulation or the cell of migration.In another embodiment, biological sample of the present invention comprises any extract or a part or whole part component of the cell or tissue sample of esophagus, such as, the original position of the esophageal carcinoma or circulation or the cell of migration.

In some embodiments, the patient being adapted to pass through method of the present invention treatment is asian ancestry or African.In some embodiments, described patient is asian ancestry and illustrates the existence of one or more EGFR biomarkers.In some embodiments, described patient is East Asia descendants.

In some embodiments, the esophageal carcinoma for the treatment of is esophageal squamous cell carcinoma (SCC).The esophageal carcinoma is generally the cancer resulting from esophagus epidermis or surperficial nexine (surfacelining).Most esophageal carcinoma falls into one or both types: squamous cell carcinoma, it is in its performance and similar to head and neck cancer on Nicotiana tabacum L. and the dependency of alcohol consumption, and adenocarcinoma, it is often relevant with Barrett esophagus (Barrett'sesophagus) medical history to gastroesophageal reflux disease.

Any suitable detection can be used to determine the histology of cancer.These tests and detection include but not limited to, the usual symptom of the esophageal carcinoma or symptom, and it includes but not limited to the adverse movement (backflow) of food by esophagus and likely oral cavity, with the chest pain that feed is irrelevant, solid or liquid dysphagia, heartburn, spit blood, hoarseness, chronic cough, singultus, pneumonia, skeleton pain, enter Endo-esophageal hemorrhage and lose weight, medical history and physical examination, imaging test, chest X-ray, computed tomography (CT) scans, nuclear magnetic resonance (MRI) scans, positron emission computerized tomography (PET) scans, bone scanning, sputum cytology, aspiration biopsy, bronchoscopy, ultrasonic in bronchus, endoscope's esophagus ultrasound, mediastinoscopy and mediastinotomy, thoracentesis, thoracoscopy, immunohistochemistry, molecules is tested, blood count, gulp down barium, ultrasonic endoscope, esophageal-gastric duodenoscopy (esophaogastroduodenoscopy) (EGD) and biopsy, or from its derivative any suitable method.

In some embodiments, anti-EGFR agent therapy can be used jointly with one or more chemotherapy, X-ray therapy, chemoluminescence therapy or targeted therapies.

In some embodiments, described chemotherapy includes but not limited to vinblastine (vinblastine), vincristine (vincristine), dactinomycin (dactinomycin), daunorubicin (daunorubicin), amycin (doxorubicin), etoposide (etoposide), mithramycin (mithramycin), paclitaxel (paclitaxel), docetaxel (docetaxel), cisplatin (cisplatin), carboplatin (carboplatin), fluorouracil (fluorouracil), folinic acid (folinicacid) and Irinotecan (irinotecan).

In some embodiments, described targeted therapies includes but not limited to bevacizumab (bevacizumab), Herceptin (trastuzumab), Erlotinib (erlotinib), Victibix (panitumumab), Sorafenib (sorafenib), infliximab (infliximab), adalimumab (adalimumab), basiliximab (basiliximab), daclizumab (daclizumab) and omalizumab (omalizumab).

In some embodiments, described X-ray therapy is used with the dosage of about 40Gy to about 80Gy.In some embodiments, dosage is about 50Gy to about 70Gy, and in some embodiments, described dosage is about 50Gy to about 65Gy.In some embodiments, described X-ray therapy is used with the dosage of about 50Gy, about 55Gy, about 60Gy or about 65Gy.

In some embodiments, the invention provides as predicting, determining, assess or monitor the method providing useful information by the treatment of the anti-EGFR agent therapy for treating esophageal carcinoma or effect.These methods are included in the presence or absence determining one or more EGFR biomarkers in the biological sample from patient, and providing about one or more EGFR biomarker presence or absence information to entity, described entity provides determination to treatment or effect or assessment based on the presence or absence of one or more EGFR biomarkers.If one or more EGFR biomarkers exist, described entity can provide the determination result that use and maybe should continue anti-EGFR agent therapy for treating.If one or more EGFR biomarkers do not exist, described entity can provide the determination result that should not use and maybe should stop anti-EGFR agent therapy for treating.

The present invention also provides test kit.Test kit of the present invention comprises at least one for measuring the reagent of one or more EGFR biomarkers and optional for using one or more EGFR biomarker results to determine the description of esophageal carcinoma therapy in biological sample.For example, the existence of one or more EGFR biomarkers indicates the respondent of anti-EGFR agent therapy, and EGFR biomarker does not exist the non-responder indicating anti-EGFR agent therapy.

Embodiment

the response of ESC to Cetuximab is determined in embodiment 1:EGFR gene amplification

Summary

Background: esophageal squamous cell carcinoma (SCC) is fatal malignant tumor (malignance) and popular especially in some areas of China, and it does not have the selection of efficient targeting therapy.Cetuximab has been approved for metastatic colorectal cancer (mCRC) and the Head and neck squamous cell carcinoma that EGFR is expressed in treatment, but not for the detection of esophagus SCC.

Method: we have evaluated Cetuximab at a series of anti-tumor activity be derived from heteroplastic transplantation (PDX) model not meeting subject esophagus SCC patient, and identify by the gene expression and oncogenic mutation analyzing (profile) these models the predictive biomarkers can determining respondent and non-responder.

Find: in 15 routine esophagus SCCPDX models of test, it is all to a certain extent to Cetuximab response, and its response rate is significantly higher than seen in other cancer types.Expressing information analysis and copy number object mutation analysis disclose response to increase with EGFR gene and high expressed directly related.This observed result prompting EGFR is crucial carcinogenic driver to this disease.Cetuximab can be effective therapeutic choice of esophagus SCC patient and EGFR gene amplification/and to express can be the predictive biomarkers of this ESC subgroup.Be necessary to carry out the indication that further clinical research helps expand Cetuximab in AC-SCC.

The esophageal carcinoma (ESC) be the most fatal cancer this, it has 5 years survival rates (the national esophageal-gastric cancer examination: in the examination of England and Wales to the nursing that the personage suffering from esophageal-gastric cancer obtains lower than 10%.Annual report London for the third time, NHS information centre; 2010).Existing two kinds of main ESC histological types: squamous cell carcinoma (SCC) and adenocarcinoma (ADC).Except operation, the therapeutic choice of standard is chemoluminescence therapy (CRT).All these has limited effect.Esophagus SCC is popular especially in some areas of China, and does not have effective targeted therapies.

Cetuximab is a kind of monoclonal antibody, and it is combined with EGFR and blocks the downstream signal that EGFR part induces and conducts.Food and medicine Surveillance Authority (FDA) approved Cetuximab is used for the treatment of transitivity CRC (the mCRC) (CiardielloFandTortoraG.NEnglJMed.2008Mar13 at colon 12/13 place without the expression EGFR of the KRAS activated; 358 (11): 1160-74), and the squamous cell carcinoma of head and neck (SCCHN) (BonnerJA, waits LancetOncol.Jan; 11 (1): 21-8), a kind of cancer types similar to ESC-SCC.The existing multinomial combination treatment about Cetuximab/chemotherapeutics is used for the II clinical trial phase of ESC in late period.(OkinesA, waits NatRevClinOncol.2011Aug for testing the activity of Cetuximab in ESC patient to have implemented a handful of test; 8 (8): 492-503).In a test, the 57 routine ESC cancer patients with CRT therapeutic alliance, wherein 48 routine ADC and 12 routine SCC.49/57 (70%) has clinical response completely after CRT, and (SafranH, waits IntJRadiatOncolBiolPhys.2008Feb1; 70 (2): 391-5).One in China comprises in the small research of 5 routine gastric cancer (GC) and 5 routine ESC-SCC, better clinical benefit has been found for ESC-SCC, but this sample number is due to too little and cannot conclude that (QiuH-j, waits JournalofOncology.2012; 16 (4): 294).SCOPE1, one group to be carried out with the ESC made a definite diagnosis and to determine use or do not use 2 groups of II/III phases of the chemoluminescence therapy of Cetuximab to test, and is just carrying out that (HurtCN, waits BMCCancer.2011 at present at UK; 11:466).Also need to test esophagus SCC Cetuximab.

The heteroplastic transplantation model (PDX) being derived from patient, when without any extracorporeal treatment, can reflect that (DingL, waits Nature.2010Apr15 for the histopathology of patient and hereditism's spectrum; 464 (7291): 999-1005; MarangoniE, waits ClinCancerRes.2007Jul1; 13 (13): 3989-98; NematiF, waits ClinCancerRes.2010Apr15; 16 (8): 2352-62; NematiF, waits Anticancerdrugs.2010Jan; 21 (1): 25-32; FichtnerI, waits ClinCancerRes.2008Oct15; 14 (20): 6456-68; AndHennesseyPT, waits PLoSOne.2011; 6 (5): e20584).It has the clinical front cancer model predictive ability of improvement and the predictive biomarkers for targeted therapies is found.Due to the extensive multiformity of cancer patient colony, the success of clinical trial depends on the target and the respondent likely with correct gene spectrum of including expression expection in a great extent, and gets rid of non-responder.The multiformity of tumor in larger PDX model set possibility potential reflection patient, and therefore by simulating Clinical Trial Model for detecting Investigational targeted drug.

Establish and be called as ESC the larger ESCPDX set of model.Be derived from the anti-tumor activity assessing Cetuximab in xenotransplantation (PDX) the model colony of the esophagus SCC patient not accepting to treat in 15 examples, and identify by the gene expression and oncogenic mutation analyzing these models the predictive biomarkers determining respondent and non-responder.Therefore these Notes of Key Data Cetuximabs can be the novel effective therapeutic choice for ESC-SCC patient (comprising the particular patient from East Asia).

Materials and methods

Implantation in patient tumor samples and immunologic injury mice.The fresh ESC-SCC tumor tissues that operation is taken out is after surgery at once for implanting the mice of immunologic injury.Other people have generally described subcutaneous implantation, and (MarangoniE, waits Clin cancer Res.2007Jul1; 13 (13): 3989-98).In brief, tumor is cut into 3x3x3mm ³fragment subcutaneous vaccination on the side of body of mice (Balb/c nude mice, 6-8 age in week, female, BeijingHFKBioscienceCo.Ltd., Beijing, China).The growth of twice pair of tumor is monitored weekly.The tumor model from these Patient Sample A set up is called as 0 generation or P0, when tumor size reaches 500-700mm ³time (1/2 long x is wide ²) serial implantation is again carried out to it, be called P1,2,3 ... (<10) generation, and implement research (analysis of pharmacology, histopathology, immunohistochemistry, cytology and molecules).The acquisition of Patient Sample A and use informed consent by the license of Ethics Committee of Beijing Tumour Hospital and patient.All processes are all aseptically implemented.The laboratory animal of all this research of participation all operates in strict accordance with the nursing of the laboratory animal of NIH and the suggestion of guide for use.

The assessment that tumor is replied Cetuximab in PDX model.When gross tumor volume reaches 100-150mm ³time, mice is randomized two groups to having similar mean tumour volume, often organizes 5.First group is carried out treating (PBS, IP injection weekly, continues two weeks) with vehicle control after grouping at once, and second group with Cetuximab (IP injection weekly, lasting two weeks, 1mg/ mice, was presented by BMS).Twice pair of tumor growth is monitored weekly, and calculates for evaluating tumor to the response of Cetuximab (in Δ T=treatment group the change of gross tumor volume and the change of gross tumor volume in Δ C=matched group) % Δ T/ Δ C value.

The mutation analysis of Model Tumor.Implement oncogene analysis of central issue with qualification sudden change in tumor.In brief, from tissue, the mini test kit of DNeasy (QIAGEN, Valencia, CA) is used to extract genomic DNA according to product description.All DNA sample are carried out pcr amplification in the reaction of 50 μ L.All detailed primer information is all described in supplemental information.In 50 μ L reactant mixtures, implement the polymerase chain reaction of 40 circulations, this mixture comprises: 100ng genomic DNA, 5 μ L10XPCR buffer, 0.2 μM of often kind of primer, 0.2mM4XdNTPs and 1 μ LTaqE.The PCR primer of amplification is carried out gel-purified and is checked order by Sanger automatic sequencer (ABI).By BioEdit software analysis data.For EGFR gene analysis of central issue, described primer is: EGFR-exons 1 9-F:5-GTGCATCGCTGGTAACATCCA-3 (SEQIDNO:1); EGFR-exons 1 9-R:5-GGAGATGAGCAGGGTCTAGAGCA-3 (SEQIDNO:2); EGFR-extron 20-F:5-CGCATTCATGCGTCTTCACC-3 (SEQIDNO:3); EGFR-extron 20-R:5-CTATCCCAGGAGCGCAGACC-3 (SEQIDNO:4); EGFR-exon 2 1-F:5-TGGCATGAACATGACCCTGAA-3 (SEQIDNO:5); EGFR-exon 2 1-R:5-CAGCCTGGTCCCTGGTGTC-3 (SEQIDNO:6).For KRAS analysis of central issue, described primer is: KRAS-exon 2-F:5-TTATGTGTGACATGTTCTAAT-3 (SEQIDNO:7); KRAS-exon 2-R:5-AGAATGGTCCTGCACCAGTAA-3 (SEQIDNO:8); KRAS-exon 3-F:5-TCAAGTCCTTTGCCCATTTT-3 (SEQIDNO:9); KRAS-exon 3-R:5-TGCATGGCATTAGCAAAGAC-3 (SEQIDNO:10); KRAS-exon 4-F:5-TTGTGGACAGGTTTTGAAAGA-3 (SEQIDNO:11); KRAS-exon 4-R:5-AGAAGCAATGCCCTCTCAAG-3 (SEQIDNO:12).PI3K-exons 1, F:5 '-CTCCACGACCATCATCAGG-3 ' (SEQIDNO:13) R:5 '-GATTACGAAGGTATTGGTTTAGACAG-3 ' (SEQIDNO:14).PI3K-exon 9, F:5 '-GATTGGTTCTTTCCTGTCTCTG-3 ' (SEQIDNO:15), R:5 '-CCACAAATATCAATTTACAACCATTG-3 ' (SEQIDNO:16), PI3K-extron 20: F:5 '-TGGGGTAAAGGGAATCAAAAG-3 ' (SEQIDNO:17), R:5 '-CCTATGCAATCGGTCTTTGC-3 ' (SEQIDNO:18).C-MET-exons 14, F:5 '-TGGGCACTGGGTCAAAGTCTC-3 ' (SEQIDNO:19), R:5 ' AACAATGTCACAACCCACTGAGGTA-3 ' (SEQIDNO:20).C-MET-exon16, F:5 '-ATTAAATGTTACGCAGTGCTAAC-3 ' (SEQIDNO:21), R:5 '-GGTTGCAAACCACAAAAGTAT-3 ' (SEQIDNO:22).C-MET-exons 17, F:5 '-GTATTCACTGTTCCATAATGAAGT-3 ' (SEQIDNO:23), R:5 ' GATGGCTGGCTTACAGCTAGTT-3 ' (SEQIDNO:24) .c-MET-exons 18, F:5 '-AACAGTAGATGCTTAGTTTATGCT-3 ' (SEQIDNO:25) R:5 '-AACAGATTCCTCCTTGTCACTT-3 ' (SEQIDNO:26) .c-MET-exons 19, F:5 '-TTCTATTTCAGCCACGGGTAAT-3 ' (SEQIDNO:27), R:5 '-ATGAAAGTAAAAGAGGAGAAACTC-3 ' (SEQIDNO:28) .c-MET-exon 21, F:5 '-CACCCTAAAGCCGAAATGCG-3 ' (SEQIDNO:29), R:5 '-CAAGGAGCAAAGAATATCGATGGC-3 ' (SEQIDNO:30) .AKT-exon 3, F:5 '-ACATCTGTCCTGGCACAC-3 ' (SEQIDNO:31), R:5 '-GCCAGTGCTTGTTGCTTG-3 ' (SEQIDNO:32) .BRAF-exons 15, F:5 '-CTCTTCATAATGCTTGCTC-3 ' (SEQIDNO:33), R:5 '-GTGAATACTGGGAACTATG-3 ' (SEQIDNO:34) .ERK-exon 2, F:5 '-ACTTTACCAACTTGCCTTCT-3 ' (SEQIDNO:35), R:5 '-TCACAACAAACCATCCCT-3 ' (SEQIDNO:36) .ERK-exon 8, F:5 '-TGCCTTACCCATAAC-3 ' (SEQIDNO:37), R:5 '-GGACCTTGAGGAACATAAT-3 ' (SEQIDNO:38).

PERK-IHC and the pEGFR-IHC dyeing of tumor slide glass.Standard immunohistochemical step is employed in this research.In brief, according to Standard histological flow process, tumor tissues is fixed in the formalin of 10% neutral buffered and is embedded in paraffin.De-paraffin and rehydrated after, 95 DEG C in the 0.01M sodium citrate solution of pH6.0 by 3 μm of thick tissue slice pretreatment 30 minutes, use the anti-human pERK (CellSignaling of rabbit subsequently, Boston, or pEGFR (Epitomics USA), Burlingame, USA) antibody staining.UltraVisionLPlargeVolumeDetectionSystemHRPPolymer (instant) test kit (LabVision, Fremont, CA) is used to detect positive staining.DAB is used as chromophoric substrate, and section is oppositely dyeed with gill hematoxylin (Gill ' sHematoxylin) (FisherScientific, FairLawn, USA).Scored with the following standard of the sample evidence of blind mode to test independently by three researcheres subsequently.Intensity is scored: 0, dye-free; 1+, minimum dyeing; 2+, moderate stain; 3+, dyes by force.The maximum region of intensity under low power lens (× 100) by the qualification of scanning tumor biopsy, and use band DP71 digital camera (Olympus subsequently, Melville, NY) OlympusBX51 microscopic system under magnification at high multiple (× 400), image is taken pictures.

CFISH analyzes.AbbottPathVysionEGFRDNA probe reagent box is used to implement FISH (double-colored) step (Abbott, DownersGrove, IL) according to the description of product.EGFR (303kb) specificity of spectrum fluorescent orange group labelling is for the EGFR gene seat on chromosome 7p12, and Chromosome Enumeration Probe (5.4kb) targeting of spectrum green fluorescence group labelling is positioned at the α-satellite DNA sequence (CEP7 in the centromere region of chromosome 7; 7p11.1 – q11.1).In brief, by the de-paraffin of FFPE section, hybridize with pepsin digestion subsequently.By the slide glass degeneration of process also with probe hybridization, resist the solution that fades oppositely to dye with 15 μ LDAPI/ subsequently and detect DAPI, rhodamine (7p12) and FITC (chromosome 7) with the OLYMPUSBX51 fluorescence microscope (OLYMPUSBX51, Japan) being equipped with single tape bandpass filter in 1000x scanning.

The determination of expressing information analysis and gene copy number.Collect fresh from the mice with tumor tumor tissues, quick freezing is also stored in-80 DEG C before for heredity and genome analysis.For gene information analysis, from freezing tissue, be separated total serum IgE according to product description Trizol (Invitrogen, Carlsbad, CA), and carry out purification with the mini post of RNeasy (Qiagen).In Bioanalyzer (Agilent) upper assessment RNA quality.The RNA sample only will with high-quality (RIN>8) according to standard step ( 3 ' IVTExpressKitUserManual, Affymetrix, P/N702646Rev.8) for the expressing information analysis design mothod on AffymetrixHG-U219 array board.The original CEL data set of all samples is by RMA algorithm standard.Probe sets intensity represents with Log (2) conversion values.For the SNP/CNV experiment using AffymetrixSNP6.0 chip, isolation of genomic DNA also uses genomic DNA tissue and blood separation test kit (Qiagen) purification according to the description of product.By standard A ffymetrix step ( genome-WideHumanSNPNsp/Sty6.0UserGuide, Affymetrix, P/N702504, Rev4) implement DNA processing and chip hybridization.Carry out quality examination to original CEL data and filter removing low interpretation rate (lowcall-rate) sample, and by PICNIC (PredictingIntegralCopyNumbersInCancer, seeGreenman etc., PICNIC:analgorithmtopredictabsolutealleliccopynumbervari ationwithmicroarraycancerdata, Biostatistics, 11 (1): 164-175, 2010) and/or PennCNV method (Wang etc., PennCNV:anintegratedhiddenMarkovmodeldesignedforhigh-res olutioncopynumbervariationdetectioninwhole-genomeSNPgeno typingdataGenomeResearch17:1665-1674, 2007) gene copy number analysis is carried out, each list of references is incorporated to by carrying stating with its entirety.For some sample, determine Relative gene copy number by qPCR.In brief, identical genomic DNA is used to the amplification by carrying out based on the quantitative PCR of SYBRGreen, and it uses EGFR Auele Specific Primer (EGFR-F:5-CATGGTGAGGGCTGAGGTGA-3 (SEQIDNO:39); EGFR-R:5-CCCCACCAGACCATGAGAGG-3 (SEQIDNO:40)).Use mammal LINE-1 reverse transcription transposon gene as reference.Chromo4 system uses OpticonMonitor3 software to q-PCR data analysis to generate initial data.Initial data is subsequently with the processing of Δ CT Relative quantification method.△ CT=(the CT value of target gene)-(the CT value of reference gene).Δ CT value is converted to intensity level (POWER (△ CT ,-2)) subsequently.By all data normalizations to there is the sample of known MET copy number to obtain relative MET copy number.

Result

ESC-SCC the large subgroup of model is replied Cetuximab.By implementing the research of clinical trial sample to the esophageal carcinoma (ESC) large group of model is carried out testing for assessment of the lateral reactivity of Cetuximab for ESC.First, the mice (Balb/c nude mice) being implanted into immunocompromised host by the tumor tissues taken out performing the operation from ESC patient by subcutaneous vaccination sets up these models.Original patient diagnosis and model pathology confirm to be shown in table 1, and more details are shown in table 2.Great majority in the model set up are ESC-squamous cell carcinoma (ESC-SCC).

Table 1. the general picture of model group

The confirmation of table 2.ESC-SCC patient diagnosis and pathology and model pathology

The second, subsequently weekly with 1mg/ mice to these models impose Cetuximab treatment, continue two weeks, to assess their sensitivity to Cetuximab.What is interesting is, this therapeutic outcome demonstrates most of ESC-SCC and replys Cetuximab with high response rate (RR): 4/10 or 40% " completely reply " (CR is defined as Δ T/ Δ C<0%); Part response (PR, 0%<% Δ T/ Δ C<50%) of 6/10; And without non-responder (NR, 50%<0% Δ T/ Δ C).The quantification of the tumor response measured by Δ T/ Δ C value is summarized in table 1.The representative anti-tumor activity of model is also shown in Figure 1.

This observed result makes us feeling pleasantly surprised due to multiple reason.First, almost do not have obtainable about the clinical information of ESC-SCC to the response of Cetuximab, and second, the RR unexpectedly high to Cetuximab and other under identical treatment condition, have evaluated the formation sharp contrast of the viewed significantly lower RR to Cetuximab of the cancer types of PDX model, these cancer types comprise colorectal carcinoma (CRC) (ChenD, Deng CetuximabresponseinAsianCRCpatienet-derivedxenograftsisp redictedbyRASpathwayactivationnotKRASmutationstatus. submit in), gastric cancer (GC) (Li etc., the observed result do not delivered) and NSCLC (YangM, Deng Squamousnon-smallcelllungcancer (NSCLC-SCC) patient-derivedxenografts (PDX) fromAsianpatienthavehighresponserate (RR) tocetuximabthanthosefromnon-SCCpatient. submit in)).The strong response prompting EGFR of many cases ESC-SCC to Cetuximab may play the part of vital role in the formation of cancer driving them.Therefore Cetuximab can be the effective therapy for ESC-SCC, particularly for asian patients, as East Asia patient.

Subsequently by implement Single dose pharmacokinetic research to EGFR intracellular signaling whether certain in these tumors of being treated by Cetuximab inactivation be studied.Single dose Cetuximab with the animal 1mg/kg of tumor is treated, and in different time points results tumor (after administration 2,6 and 24 hours).By the pEGFR that IHC staining examine is organized, and pERK and pAKT, two crucial downstream signal conduction biomarkers.The representative example that ES110IHC analyzes is shown in Fig. 2 A (image) and 2B (mark quantification).This result clearly shows the reduction of all these intracellular signaling biomarkers.

EGFR gene amplification and/or process LAN are showing the ESC-SCC replied the most by force seemingly strong carcinogenic driver in subgroup.Although the ESC-SCC model of nearly all test all confirms response at present, great changes have taken place for the degree of described response, and therefore show that hereditary variation in a model may control described response.Therefore the heredity and genome biomarker that may control the response observed in these models are studied.Because EGFR is therapeutic target for respondent and obvious carcinogenic driver, be therefore first devoted to study the EGFR of these models.

Use IHC, one is normally used method clinically, confirms the expression of EGFR.Result shows that all ESC-SCC express EGFR at protein level.Next, AffymetixHG-U219 gene microarray analysis is used to be studied in the expression of mRNA level in-site to EGFR.What is interesting is, in all these models, response degree all relevant to the level of EGFR (table 1 and Fig. 3).Because the more high activity via the more EGFR of high expressed may drive carcinogenic conversion in these tumors, can Tumor suppression growth by the inactivation of Cetuximab, therefore this observation seems reasonable.

In addition, AffymetrixSNP6 and/or qPCR is also used to be studied expressing genetic flaw behind at higher EGFR by the change first detecting gene copy.What is interesting is, all respondents/EGFR high expressors has corresponding EGFR gene amplification (table 1 and Fig. 3).The amplification of this observed result prompting EGFR gene may be crucial carcinogenic driver and be the potential practical biomarker for predicting the response to Cetuximab in ESC-SCC in respondent.For confirming gene amplification further, implementing EGFR-fluorescence in situ hybridization or FISH, a kind of clinical assays of practicality, assessing the EGFR gene amplification state of all these models.FISH data confirm observed result seen by SNP6 really.For example, Fig. 3, bottom diagram, describes GA110FISH and analyzes, clearly show that the amplification (Fig. 4, left figure) of EGFR.The FISH accepted clinically makes to become possibility to the exploitation with diagnosis of Cetuximab therapy clinically.

Important problem is whether the primary tumor in PDX source also has the homologous genes amplification found in a model, or more high expressed.For this reason, further the sample of this patient tumor samples and test is analyzed jointly.In tested Patient Sample A, confirm amplification.Fig. 4, right figure, demonstrate the gene amplification that original Patient Sample A also has EGFR.

In addition, comprise KRAS, EGFR, AKT, c-met and BRAF etc. to the multiple common oncogene of activated mutant also to analyze.Ironically, ignore the response level to Cetuximab, the model tested, in the oncogene of test, comprises KRAS, BRAF, AKT, PI3KC, c-met, c-kit etc., does not show any activated mutant (table 1).

Discuss

These researchs clearly demonstrate the ESC-SCC set up from Chinese patients model is replied Cetuximab mostly to a certain extent.The degree of response is relevant with copy number to the gene expression of EGFR.There is gene amplification significantly better to Cetuximab response with those of rising expression.Therefore these data show that Cetuximab can be the effective therapeutic choice for ESC-SCC patient, particularly from the patient in East Asia.In addition, these data also support EGFR gene amplification and can be used as the Cetuximab respondent of predictive biomarkers for estimating in ESC-SCC patient.The EGFRFISH increased for EGFR gene measures conventional for adjoint diagnosis that is clinical and ESC-SCC treatment.Therefore, such expection test can easily be implemented.Really accreditation promotes that Cetuximab is used for the treatment of the supervision license of the purposes of ESC-SCC patient clinically, and it provides the first targeted therapies for ESC-SCC patient.

ESC-SCC the response of model to Cetuximab depends on high expressed and the high copy number of EGFR, with NSCLC and CRC expressed and copy number remarkable effect is lower (ChenD, Deng, CetuximabresponseinAsianCRCpatient-derivedxenograftsispr edictedbyRASpathwayactivationnotKRASmutationstatus, insubmissionandYangM, Deng Squamousnon-smallcelllungcancer (NSCLC-SCC) patient-derivedxenografts (PDX) fromAsianpatientshavehighresponserate (RR) tocetuximabthanthosefromnon-SCCpatients, insubmission) form sharp contrast.In ESC-SCC, EGFR process LAN (gene copy number of rising)-Cetuximab response relation is similar to Her2 process LAN in GC (gene copy number of rising)-trastuzumab (trastuzumab) response relation.This similarity provide for the Cetuximab therapy for ESC-SCC with diagnosis clear feasible exploitation and supervision approach (developmentandregulatorypath), as trastuzumab with diagnose.

In a word, in the esophagus SCCPDX models of 15 example tests, it is to a certain extent all to Cetuximab response, with significantly higher response rate (RR) compared with those discoveries in other cancer types.Express profile analysis and copy number mutation analysis disclose described response increase with EGFR gene and high expressed directly related.These data show that EGFR is the crucial cancer driver for this disease.Cetuximab is the therapeutic choice for esophagus SCC patient, and EGFR gene amplification/expression is the predictive biomarkers for this ESC subgroup.

Foregoing teachings merely illustrates principle of the present invention.Those skilled in the art can design different schemes, although these schemes are not clearly set forth or show herein, embody principle of the present invention, and will comprise within the spirit and scope of the present invention.In addition, the concept of Push Technology that all embodiments described herein and conditional language are mainly intended to make reader understanding's principle of the present invention and are proposed by inventor, and should not be understood to the restriction of embodiment and the condition that these are specifically recorded.

In addition, whole statements of principle described herein, aspect and embodiment of the present invention and specific embodiment thereof are all intended to contain equivalent in its result and functionally.In addition, these equivalents are intended to comprise the equivalent that equivalent known at present and future development go out, any element of the enforcement identical function namely developed, and regardless of structure.Therefore protection scope of the present invention is intended to be not limited to the shown exemplary with describing herein.Protection scope of the present invention and spirit are embodied by the claim of adding further.

Claims (21)

1., for treating a method for the esophageal carcinoma in patients, it comprises the anti-EGFR agent therapy described patient being used to effective dose.

2. the process of claim 1 wherein that described patient has one or more EGFR biomarkers.

3. for treating a method for the esophageal carcinoma in patients, it comprise detect one or more EGFR biomarkers presence or absence and when one or more EGFR biomarkers exist with anti-EGFR agent therapy for treating patient.

4. the method for the identification of respondent and/or non-responder patient, it is included in the presence or absence detecting one or more EGFR biomarkers in the biological sample from the patient suffering from the esophageal carcinoma, wherein the existence of one or more EGFR biomarkers indicate the respondent of anti-EGFR agent therapy and EGFR biomarker there is not the non-responder indicating anti-EGFR agent therapy.

5. one kind for determining the method for the therapeutic scheme being used for the treatment of the esophageal carcinoma in patient in need, comprising from suffer from the esophageal carcinoma patient biological sample in detect the presence or absence of one or more EGFR biomarkers, wherein the existence of one or more EGFR biomarkers indicate the respondent of anti-EGFR agent therapy and EGFR biomarker there is not the non-responder indicating anti-EGFR agent therapy, and when one or more EGFR biomarkers exist with patient described in anti-EGFR agent therapy for treating.

6. one kind for changing the method for the therapeutic scheme of anti-EGFR agent therapy, it is included in the presence or absence that detects one or more EGFR biomarkers in the biological sample from the patient accepting anti-EGFR agent therapy and changes therapeutic scheme based on the existence of one or more EGFR biomarkers in described biological sample, wherein continues described therapy when one or more EGFR biomarkers exist.

7. a selection suffers from the patient of the esophageal carcinoma for the method with anti-EGFR agent therapy for treating, it is included in the presence or absence detecting one or more EGFR biomarkers in the biological sample from described patient, wherein the existence of one or more EGFR biomarkers indicate the respondent of anti-EGFR agent therapy and EGFR biomarker there is not the non-responder indicating anti-EGFR agent therapy, and select those patients that there are one or more EGFR biomarkers to be used for the treatment of anti-EGFR agent therapy.

8. the method for any one of claim 2-7, wherein said EGFR biomarker is selected from the amplification of EGFR gene, the expression of EGFR, EGFRRNA level, the EGFR of constitutive activity, activity of EGFR, EGFR pathway activation or EGFR approach intracellular signaling.

9. the method for claim 8, the amplification of wherein said EGFR gene increases with the predetermined EGFR gene copy number object copied compared with number with reference to EGFR gene.

10. the method for claim 9, wherein said EGFR gene copy number is selected from least 3,4,5 and 6 or more copies.

The method of 11. any one of claim 1-11, wherein said patient is asian ancestry (Asiandescent).

The method of 12. any one of claim 1-11, the wherein said esophageal carcinoma is esophageal squamous cell carcinoma.

The method of 13. any one of claim 1-12, wherein said anti-EGFR agent therapy is anti-egfr antibodies therapy.

The method of 14. any one of claim 1-12, wherein said anti-EGFR agent therapy is the therapy of Cetuximab, its biological analog or derivant.

The method of 15. any one of claim 1-12, wherein said anti-EGFR agent therapy and one or more chemotherapy, X-ray therapy, chemoluminescence therapy or targeted therapies are used jointly.

The method of 16. claim 15, wherein said chemotherapy is selected from: vinblastine (vinblastine), vincristine (vincristine), dactinomycin (dactinomycin), daunorubicin (daunorubicin), amycin (doxorubicin), etoposide (etoposide), mithramycin (mithramycin), paclitaxel (paclitaxel), docetaxel (docetaxel), cisplatin (cisplatin), carboplatin (carboplatin), fluorouracil (fluorouracil), folinic acid (folinicacid) and Irinotecan (irinotecan).

The method of 17. claim 15, wherein said targeted therapies is selected from: bevacizumab (bevacizumab), Herceptin (trastuzumab), Erlotinib (erlotinib), Victibix (panitumumab), Sorafenib (sorafenib), infliximab (infliximab), adalimumab (adalimumab), basiliximab (basiliximab), daclizumab (daclizumab) and omalizumab (omalizumab).

18. 1 kinds for determining, assessing or monitoring anti-egfr antibodies therapy provides the method for useful information to the treatment of the esophageal carcinoma or treatment effect, it is included in the presence or absence determining one or more EGFR biomarkers in the biological sample from patient, and providing one or more EGFR biomarker presence or absence determination results described to entity, described entity provides determination to treatment or effect or assessment based on the presence or absence of one or more EGFR biomarkers.

19. 1 kinds of test kits, it comprises reagent for measuring one or more EGFR biomarkers in biological sample and the optional description for using described measurement, the existence of one or more EGFR biomarkers wherein said indicate the respondent of anti-EGFR agent therapy and one or more EGFR biomarkers there is not the non-responder indicating anti-EGFR agent therapy.

The method of 20. claim 7, wherein said method comprises the protein expression level detecting EGFR.

The method of 21. claim 20, wherein said protein expression level is determined by immunohistochemistry (IHC), immunoblotting, Western Immuno dyeing, Western Immuno precipitation, immunoelectrophoresis, immunoblotting, BCA algoscopy, spectrophotography, mass spectrography or enzyme assay.